Immunochemical assays in pesticide analysis

27 Pages·0.184 MB·English

by  Dankwardt A.

1
IMMUNOCHEMICALASSAYSINPESTICIDEANALYSIS
Immunochemical Assays in per trillion to lower parts per billion range.A lot of sam-
ples can be analyzed within a short time, while only low
Pesticide Analysis
samplevolumesarenecessary.Inmanycases(water,some
liquid food samples) no extraction step and no cleanup
arenecessary.Notallassaysarecompletelyspecifictoone
AndreaDankwardt
single compound. Cross-reactivities of the Abs with hap-
SensionGmbH,Augsburg,Germany
tenssimilartotheanalytecanbeobserved.Insomecases,
matrix effects may occur, especially with soil or colored
food extracts. Therefore, validation of the assays for the
matrixofinterestshouldbecarriedout.AsIAsareusually
1 Introduction 1
targeted at a single analyte or a group of analytes, multi-
2 Antibodies 2
analyte approaches using Ab arrays or a combination of
2.1 Antibody Structure 2
immunochemical techniques with liquid chromatography
2.2 Antibody Production 3
(LC)arepursued.
2.3 Immunogens 3
3 Immunoassay Types 5
3.1 Assay Formats 5
1 INTRODUCTION
3.2 Labels 5
3.3 Solid Phases 6
Interestinimmunochemicalassaysforthedetermination
4 Properties of Competitive Immunoassays 6
of pesticides has been steadily increasing. IAs are now
4.1 Dose–Response Curve 6
commonly applied for the analysis of contaminants
4.2 Quality Control 7
in water, soil, food and body fluids..1–9/ The first
4.3 Cross-reactivity 8
immunologicalexperimentshadalreadybeencarriedout
4.4 Sample Preparation and Matrix
as early as the late eighteenth century when Edward
Effects 8
Jenner, an English physician, used cowpox to prevent
5 Application to Environmental Samples 9
infection with smallpox. Based on these studies Louis
5.1 Water 9
Pasteur developed the use of attenuated strains of
5.2 Soil 13
microorganisms for successful vaccinations. Emile Roux
5.3 Food 13
andAlexandreYersinthenfoundthatimmunityiscaused
5.4 Biomonitoring 14
by soluble compounds of microorganisms, which they
6 New Developments 14 calledtoxins.Thesetoxins inducespecificcompounds in
Acknowledgments 16 theimmunizedanimal,whichwerenamed‘‘antitoxins’’by
Emil von Behring and Shibasaburo Kitasato (1890) and
Abbreviations and Acronyms 16
arenowcalledAbs.TheAb‘‘generating’’compoundsare
Related Articles 16
knownasAgs.
References 16 AroundtheturnofthecenturyitwasshownthatAbs
arenot only produced against microorganisms and their
toxins,butalsobyothersubstancessuchasmilk,protein
or plant-derived toxins. Paul Ehrlich was the first to
Immunochemical assays (immunoassays, IAs) are bio-
carry out quantitative studies on Ag–Ab interactions.
chemicalassayswhichworkaccordingtothelawofmass
The great interest in this field led to the first book
action. They are based on the recognition of an antigen
on immunochemistry, published by Svante Arrhenius in
(Ag) or a hapten by antibodies (Abs). Abs are serum
1907..10/KarlLandsteineralsobelongstothepioneersin
glycoproteins of the immunoglobulin (Ig) class and are
immunochemistry.Hesystematicallyusedsmallartificial
producedbythevertebrateimmunesystemagainstforeign
molecules, which he called haptens, coupled to a carrier
materialofhighmolecularmass.Theresultofthebinding
molecule for immunization. In 1923 Heidelberger and
reaction between the Ab and an analyte is usually made
co-workersfoundpolysaccharidestobeantigenicaswell.
visiblebymeansofenzymatic,chemiluminescent,fluores-
StudiesbyRodneyPorter(1959)andGeraldEdelman
cent or radioactive markers. According to the label used (1961) have provided the chemical structure of the Ab
IAs can be classified into enzyme immunoassays (EIAs), molecule.TheenormousvarietyofAbswasexplainedby
radioimmunoassays (RIAs), fluorescence immunoassays FrankMcFarlaneBurnetin1957, basedonahypothesis
(FIAs)orchemiluminescentimmunoassays(CLIAs).The ofNielsJernedatingfrom1955,thenowwidelyaccepted
measuring range of most IAsfor pesticides is in the parts clonal-selection theory. It describes each Ab-producing
EncyclopediaofAnalyticalChemistry
EditedbyRobertA.Meyers.(cid:211) JohnWiley&SonsLtd,Chichester.ISBN0471976709
2
PESTICIDES
cell as carrying on its surface only one type of Ab
as a receptor. The binding of a respective Ag to this
Occupancy of antibody binding sites by analyte
receptorleadstoaclonalexpansionofthiscellandtothe
maturationofAb-producingcells.
Immunochemical methods have their origin in the
medicalfield.ThefirstIA,aRIAforthequantificationof Occupied Unoccupied
insulininserum,wasdescribedbyYalowandBerson..11/ binding sites
Later, radiolabels were replaced by enzymes in EIAs
by Engvall and Perlmann.12/ and Van Weeman and Competitive Measurement of
Schuurs..13/ Since then radiolabels have obtained broad assays unoccupied
application in medical diagnostics and environmental binding sites
analysis.

IAs belong to the most common methodology in the (a) Immobilized antibody (b) Immobilized coating
field of immunoanalysis. Even though Abs are (still) conjugate
produced by a biological process, IAs are nevertheless
chemical analytical procedures. The basic principle
applyingtoallimmunoreactionsisbaseduponthelawof
mass action. In the equilibrium reaction between a free
Agorahapten,suchasapesticide,andtheAbformingthe
hapten–AbcomplexHAb(Dboundhapten),represented
Inverse relation between Inverse relation between
byEquation(1),
antibody-bound tracer and coating conjugate-bound
analyte concentration labelled antibody and
HCAbDHAb .1/ analyte concentration
theaffinityconstantKdeterminestheconcentrationratio
betweentheboundhaptenandthefreereactionpartners, Antibody Iamntmiboobdiylized Lanatbibeoleddy
Equation(2):
Analyte Immobilized Labeled
[HAb]
K D mol(cid:0)1 .2/ (hapten) analyte analyte
[H][Ab]
A low detection limit (DL) in an IA therefore requires
Figure1 Principle of the competitive IA. In the first format
a high affinity of the Ab toward the analyte, which is withimmobilizedAb(a)theplatesarecoatedwithAb.Analyte
expressed by a high affinity constant. For further details andenzyme-labeledanalytecompetefortheAbbindingsites.In
refertoHocketal..14/ thesecondformat ahapten–proteinconjugateisimmobilized
in the solid phase(b). This protein conjugate and the free
IAs are based upon the measurement of Ab binding-
analyte compete for the binding sites of the Ab in solution.
siteoccupancybytheanalyte(Figure1).Thisreflectsthe
(Reproduced from Dankwardt and Hock.7/ with permission
analyte concentration in the sample. Since the binding fromFoodTechnologyandBiotechnology.)
reactiondoesnotproduceasignalwhichcanbedetected
by simple means, various markers, e.g. radioactivity,
enzymes, or fluorescence, are employed for the detec- of the Ig class produced by the immune system against
tion of the immunoreaction (see section3.2). However, foreign material such as pathogens or xenobiotics, and
moresophisticatedtechniqueslikesomeimmunosensing bindthetargetsubstancewithhighselectivityandaffinity.
methodsdonotrelyonalabel(seesection6). Although there are five distinct classes of Ab in most
higher mammals (IgA, IgD, IgE, IgG, IgM) IgG makes
up approximately 80% of the total Ig in human serum.
MostIAsrelyuponIgGasthemajorIg.
2 ANTIBODIES
The basic structure of an Ab molecule is shown in
Figure2. It consists of two identical heavy (H) chains
2.1 AntibodyStructure
and two identical light (L) chains stabilized and linked
Immunochemical analysis is based upon the specific by inter- and intrachain disulfide bonds. The H- and
reaction between an Ab and its corresponding Ag or L-chainsareorganizedintovariableandconstantregions.
hapten.Absarepartofthevertebratedefensesystem(for The Ag binding site (combining site) is formed by the
moredetailsrefertoimmunologytextbooks,forexample association of parts of the variable regions of the H-
Golub.15/ and Roitt.16/). They are serum glycoproteins and L-chains, located at the amino terminal end. The
3
IMMUNOCHEMICALASSAYSINPESTICIDEANALYSIS
Heavy chain FR1 Antigen binding site of interactions such as hydrophobic, ionic, H-bonding,

Light chain FFRRF 4 R32 CDR1 bpi-npdeinlegcetrnoenrgiynt(errealacttiivoen,afafinnditvyaonfdtheerAWba)ailnscfroeracseess.wTihthe
VH CDR2
VL CDR3 the number of specific chemical interactions between
the analyte and the amino acid residues in the Ab
combining site. Therefore, the selectivity and sensitivity
CH1 ofanIAiscontrolledbythenatureoftheAg–Abbinding
CL VL VH process.
2.2 AntibodyProduction
VL VH CH2
Fv
Ab production is conveniently carried out in warm
Carbohydrate bloodedanimals,e.g.rabbits,sheep,miceorchickens..17/
Polyclonal antibodies (pAbs) are obtained from the
CL CH1 CH3 CL VH serum and comprise a mixture of different Ab popu-
lations.Monoclonalantibodies(mAbs)consistofasingle
monospecific Ab population. These Abs are produced
Fab Antibody scFv in cell culture by a single hybridoma cell derived from
the fusion of B-lymphocytes with myeloma cells..18/ The
Figure2 StructureofIgGAbsandtheirfragments(modified hybridoma cells can then be propagated almost indefi-
afterHocketal..17/).scFvistherecombinantantibodyfragment,
nitely in culture and will continue to produce the Ab of
asinglechainfragmentcontainingonlythevariableregion,FR
the lymphocyte parent. Since an individual lymphocyte
istheframeregion,V isthevariableregionoflightchain,V
L H
isthevariableregionofheavychain,C istheconstantregion produces only a single Ab type, all of the Ab molecules
L
onthelightchain,CH ,CH ,CH isaconstantregiononthe produced by a hybridoma cell line derived from a sin-
1 2 3
heavychain. gle hybrid cell are identical and have the same binding
properties. Therefore, the hybridoma technology guar-
variable regions of both chains are organized into three antees the unlimited production of mAbs with constant
hypervariable or complementarity determining regions characteristics..19/ Owing to the great effort involved in
(CDRs) separated by four framework regions. The mAb production many IAs still employ pAb. A third
greatest amino acid sequence variation occurs within possibility for creating Abs has emerged, recombinant
the CDRs whereas the framework regions are more antibody(rAb)techniques.Here,Iggenescanbecloned,
conserved. It is assumed that the association of the introduced and expressed in inexpensive and relatively
CDRregionsformsthecombiningsite.Thelowerpartof simple host systems..20;21/ Although several nonmam-
the molecule, the Fc (antibody fragment containing the malian host systems (yeast, plant and insect cells) have
crystallizablefragment)isresponsibleforsomeimportant been used to produce rAbs, the most common vehicle
biologicaleffectorfunctionssuchascomplementfixation is Escherichia coli..22;23/ The main properties of pAbs,
andisnotnecessaryforAgorhaptenbinding.Itcontains mAbsandrAbsarelistedinTable1.
the last heavy chain domains. The whole of the Ig
molecule or Ab fragments, F(ab) and Fab (antibody
2 2.3 Immunogens
fragments containing the antigen binding site(s)) can be
usedinIAs. Mostpesticidesareoflowmolecularmassandtherefore
A substance that after injection into the body of a arenot ordinarily antigenic. Theyhave to be coupled to
vertebrateinducesaspecificAbsynthesis,iscalledanAg. a carrier molecule, usually a protein, in order to induce
Agsareprincipallymacromolecules,forinstanceproteins, anAbresponseinthevertebrateimmunesystem..14/The
polysaccharidesornucleicacids.Syntheticpolymersalso site of coupling to the carrier, the coupling procedure
belongtotheantigens,i.e.theycanbeusedas,oractas, as well as the number of haptens bound to one
Ags. Small molecules (haptens) such as pesticides have carrier molecule can be of major importance for the
tobecoupledtoamacromolecularcarriertoelicitanAb sensitivity and the selectivity of the resulting Ab (for
response(seesection2.3).TheabilityofanAbmolecule reviews refer to Erlanger,.24;25/ Goodrow etal..26/ and
to bind an Ag or a hapten specifically is controlled Szurdokietal..27/).
by structural and chemical interactions between the The protein carriers used in various laboratories
ligand and the Ab at the combining site. The Ag–Ab include globulin fractions, serum albumins of different
interaction is reversible and does not involve formation species, hemocyanin, ovalbumin, thyroglobulin, and fib-
ofcovalentbonds..16/ Thebindingisaresultofavariety rinogen.Alsononproteinaceouscarriershavebeenused
4
PESTICIDES
Table1 Propertiesofpolyclonal,monoclonalandrecombinantAbs
Properties pAb mAb rAb
(Abfromserum) (Abfromhybridomacells) (Abproducedbygenetechnology)
Supply Limitedandvariable Unlimitedproductionpossible Unlimitedproductionpossible,
immunizationnotmandatory
Uniformity Changingpropertieswith ConstantpropertiesofamAb ConstantpropertiesofarAb,canbe
differentseraandbleedings changedbygeneticmanipulations
Affinity MixtureofAbwithdifferent Uniformlyhighorlow,canbe Uniformlyhighorlow,canbe
affinities,affinityoftenhigher selectedbytesting selectedbytestingandcanbe
withpAb modified
Cross-reactivity Resultsfromdifferent Different,dependentuponthe Different,dependentuponthe
selectivitiesandlowaffinity individualAb individualAb,canbemodified
interactions
Classesand Typicalspectrum Onedefinedisotype Different,dependingonmolecular
subclasses design
DemandsonAg Highpurityrequiredforspecific ImpureAgsormixtureofAgs ImpureAgsormixtureofAgscan
antisera canbeusedforimmunization, beusedforimmunization,pure
pureAgsnecessaryfor Agsnecessaryforscreening,
screening immunizationnotmandatory
Costs Low High Onceestablished,low
suchasliposomesordextran..28;29/Keyholelimpethemo- determinant structures..26/ Usually C –C spacers are
3 6
cyanin (KLH), a protein from mollusks, is often viewed used; if the spacer is too long, it may bend back to the
asasuperiorcarrierbecauseitisforeigntothevertebrate carrier and the hapten will not be properly exposed.
immunesystem..30/ StrategiesforIAhaptendesignforthetriazine,arylurea
Anotherimportantissueconcernstheoptimalnumber and chloroacetanilide herbicides have been summarized
of haptens bound to the carrier protein (i.e. optimal byGoodrowetal..26/
epitopedensity).Highlysubstitutedcarriersusuallylead The functional groups of the hapten govern the
to the best results. For bovine serum albumin (BSA) selection of the method to be used to conjugate the
molar ratios of 10:1 to 20:1 (hapten:carrier) are hapten to the functional groups of the carrier. The
desirable;forlargermoleculessuchashemocyanin,ratios functional groups of the protein carrier available for
of800:1to1000:1shouldbeobtained..17/However,very attachment of thehaptens arethecarboxyl groupof the
highratiosmayreduceimmunogenicitybecauseofeither Cterminalandoftheasparticandglutamicacidresidues,
the changes in tertiary structure of the protein caused theaminogroupoftheNterminalandthelysineresidues,
by masking of the essential free amino groups or the the imidazol and phenolic functions of the histidine and
removal of critical determinant sites on the carrier by tyrosineresidues,respectively,andthesulfhydrylgroupof
haptenicblocking. cysteineresidues.Generalproceduresforthepreparation
For the production of pAbs the purity of the hapten ofconjugatescanbefoundinErlanger..24;25/
used to prepare an immunoconjugate should be as After coupling, characterization of the conjugates can
high as possible. After synthesis of haptenic substances, be carried out (see Erlanger.25/). Generally, the hap-
closely related substances may be present in small tenic groups have an absorbance spectrum that can be
quantities, leading to the production of nonspecific Abs differentiated from the protein carrier. Elemental anal-
and, consequently, to unwanted cross-reactivities of the ysis for the chlorine content can be carried out for
antisera.ThisisnotaproblemwithmAb,becauseasingle some triazine conjugates. A more direct procedure is
celllineproducingonlyonekindofAbwiththedesired theincorporationofsomeradioactivehapteninthecon-
propertiescanbeselected. jugation procedure. Another approach is quantitating
Another important consideration is the point of the change in free amino groups as a result of conjuga-
attachment on the hapten. Ab specificity is directed tion.Arecentlyappliedtechniqueisthedeterminationof
primarily at the part of the hapten molecule farthest haptendensitybymatrix-assistedultravioletlaserdesorp-
away from the functional group that is linked to the tion/ionization mass spectrometry (MALDIMS).31/ and
proteincarrier..25/Evenbetterspecificitycanbeobtained electrospray ionization mass spectrometry (ESIMS)..3/
with conjugates in which the hapten is coupled to the Application of energy-minimized molecular modeling
carrierviaaspacer,therebygivingmuchbetterexposure methods to hapten design will help to choose the best
of the hapten on the surface of the carrier. The spacer derivatives and conjugation methods for successful Ab
should be attached as far as possible from the unique production..32/
5
IMMUNOCHEMICALASSAYSINPESTICIDEANALYSIS
3 IMMUNOASSAYTYPES withthetracerforbinding..37/Whiletheseassaysareeasy
tocarryoutandverysuitableforautomation,theyusually
3.1 AssayFormats show a lower sensitivity than EIAs, e.g. for simazine a
DLof5m gL(cid:0)1wasobserved..37/
For low-molecular-mass analytes (haptens) such as
Noncompetitive assays can only be applied for high-
pesticides in solution, competitive tests have to be
molecular-mass analytes with more than one antigenic
employed,usinglimitingAbconcentrations.Thetestscan
determinant (i.e. Ag) or low-molecular-mass analytes
beperformedashomogeneousassayswithoutseparation
(haptens)boundtoasolidphase,exposingtheantigenic
ofthereactants,.33/butmorecommonareheterogeneous
determinant. They work with an Ab excess. Noncom-
tests where unreacted reagents are removed before
petitive IAs have been employed for the detection of
evaluation. Twodifferent formats areavailable, (1)with soil-bound pesticides..38;39/ In this case the soil particles,
immobilized Ab (Figure1a) and (2)with immobilized
towhichthepesticideresidueshavebound,formthesolid
coatingconjugate(Figure1b).Invariant(1)analyteand
phase,andtheresiduescanbedetectedbyalabeledAb
alabeledanalyte(tracer)competeforthefreeAbbinding
specifictotheanalyte.
sites. After removal of unbound reactants the bound
tracer yields a signal that is inversely proportional to
the analyte concentration. The variant (2)employs an 3.2 Labels
immobilizedhapten-carrierconjugateonthesolidphase
to which analyte and Ab are added. The Ab binds to Depending on the label, IAs are classified in different
the free analyte or to the immobilized hapten according groups. Radioisotopes are used in RIAs, enzymes in
to the concentration of the reactants. If a labeled Ab is enzyme-linkedimmunosorbentassays(ELISAs)orEIAs,
used,theamountofAbboundtothesolidphasecanbe fluorophores in FIAs or PFIAs and chemiluminescent
directly determined after a washing step. Alternatively, compounds in CLIAs. Additional types of IA exist,
a secondary labeled Ab may be used to detect the Ab but are not very common in pesticide analysis. A
whichhasboundtothesolidphase.Thesignalisinversely more detailed description of these IAs can be found
proportionaltotheamountoffreeanalyteinthesample. inGosling..40/
VerysensitivecompetitiveIAshavebeendevelopedwith EIAs are most commonly used in pesticide analysis
DLsbetween1and50ngL(cid:0)1,forexampleforthetriazines as they avoid the necessity of working with radioactive
andureaherbicides..34–36/ material and low DLs can be reached. Simple and
An example for a homogeneous assay system is cheap photometers which give an extremely rapid
thepolarizationfluoroimmunoassay(PFIA).PFIAmea- measurement capability and long-lasting stability of the
sures the increased polarization of fluorescence when a coloredproductafterthereactionhasstoppedmakeEIA
fluorophore-labeledhapten(tracer)isboundbyaspecific superiortofluorimetryorluminometry,eventhoughwith
Ab,andthedecreasedsignalwhenfreeanalytecompetes these methods lower DLs may be reached..33/ Enzymes
Table2 EnzymesystemscommonlyusedforEIAs
Enzyme Source Molecular pH Colorimetric Fluorometric Luminometric
weight optimum substrates substrates substrates
Alkaline Calfintestine 100000 9–10 p-Nitrophenyl- 4-Methylumbelliferyl- Adamantyl-1,2-
phospha- phosphate phosphate dioxyethane
tase Phenylphosphate-
substituted
dioxyethane
b-Galacto- Escherichiacoli 540000 6–8 o-Nitrophenyl-b-D- 4-Methylumbelliferyl- –
sidase galactopyranoside b-D-galacto-
Chlorophenolic pyranoside
red-b-D-galacto-
pyranoside
Peroxidase Horseradish 40000 5–7 2,20-Azino-di(3-ethyl- p-Hydroxyphenyl- Luminol
benzthiazolinesulfonic aceticacid
acid-6)(ABTS)/H O p-Hydroxyphenyl-
2 2
3,30-5,50-Tetramethyl- propionicacid
benzidine(TMB)/H O
2 2
o-Phenylendiamine
(OPD)/H O
2 2
6
PESTICIDES
Table3 SolidphasesusedforEIAs
Material Form Binding Capacity
Polystyrene Microtiterplates,tubes,pins, Noncovalent 250–500ngcm2
beads
Polyethylene Tubes Noncovalent ca.300ngcm(cid:0)2
Polypropylene Microtiterplates Noncovalent ca.300ngcm(cid:0)2
Polyvinylchlorideand Microtiterplates,membranes Noncovalent ca.300ngcm(cid:0)2(plates)
similar
Polycarbonate Beads,membranes Noncovalent ca.300ngcm(cid:0)2(beads)
Nitrocellulose Microtiterplates,membranes Noncovalent ca.100m gcm(cid:0)2
ProteinAcoated Microtiterplates,beads Noncovalent 20mgmL(cid:0)1(forAbonly)
Activatedpolymer,with Microtiterplates,beads Covalent,using 2(cid:2)1013–1(cid:2)1014reactive
aminoorcarboxylgroups bifunctionalreagents sites/cm2
Magnetic Beads Dependsonthe Varies
surfaceofthebeads
commonlyusedaslabelsinheterogeneousEIAarelisted covalentbinding. Thosesolidsupportscontainaminoor
inTable2. carboxygroupsonamodifiedsurfacethroughwhichthe
The following requirements are necessary for the use immunoreagentscanbeboundbywater-solublecarbodi-
ofanenzymeasamarker: imidesorbifunctionalreagentssuchasglutaraldehyde.
Other solid-phase supports for IAs are membranes.
(1) high specific activity (turnover number) of free They can be used for dip sticks, which are incubated
enzymeandafterlabeling, for a short time in the solution.36/ or for dot blots and
(2) availability of soluble, purified enzyme at low cost immunofiltration tests. Here the reactants are filtered
andreproduciblequality, through the membrane..43;44/ The test principle is the
(3) high stability in free and conjugated form under same as for the microtiter plate tests but the reaction
storageandassayconditions, time is much shorter owing to the high surface area of
(4) presence of reactive groups for covalent linkage to the membrane and the short distance between reaction
hapten, partners. Application of remission measurements yields
(5) simpleandgentleconjugationmethods, aproportionalrelationshipbetweenanalyteandremitted
(6) inexpensive and stable nontoxic substrates with light.Byusingapocketreflectometer,thisset-upisideally
formationofstablechromogenic,fluorogenicand/or suitedforfield-monitoringpurposes..45;46/
chemiluminogenicproducts.
3.3 SolidPhases 4 PROPERTIESOFCOMPETITIVE
IMMUNOASSAYS
IAs are mainly carried out in 96-well polystyrene,
polyethylene, polypropylene or polyvinyl microtiter
4.1 Dose–ResponseCurve
plates, owing to the easy separation of the reactants
in a washing step, but polystyrene tubes, beads or pins In IAs the signal produced is inversely correlated to
are also available (Table3). The plastic plates are of the analyte concentration in the sample (Figure3a).
comparativelylowbindingcapacityandlowsurfacearea The typical dose–response curve is of sigmoidal shape
to volume ratio. High-binding supports include agarose when the signal is plotted versus the logarithm of
and cellulose. Particulate solid phases are very efficient, the analyte concentration. A linear range is obtained
because they become scattered throughout the reaction around the middle of the test (IC , middle of assay,
50
mixture and have a much higher surface area to vol- concentration of analyte that causes 50% inhibition),
ume ratio..41/ For example, many chemically different which should be used for determinations. Within this
beads are available (e.g. polystyrene, latex, polycarbon- working range, the change in absorbance is linearly
ate and copolymer beads). Immunological reagents are correlated to the analyte concentration. The linear part
bound to the beads in a similar manner as they are to of the curve is confined by the upper and lower limits
microtiterplates.Separation ofboundandfreereagents of quantification. These are the cut-off values above
occurs by washing and centrifuging. IAs using magnetic or below which quantitative results can be obtained
beads employ a magnet for the separation step..42/ Abs with a stated relative precision, or specified degree of
and Ags may be immobilized to some solid phases via confidence in real samples..47/ The experimental errors
7
IMMUNOCHEMICALASSAYSINPESTICIDEANALYSIS
increase toward these limits. Consequently, the most
precise measurements are obtained in the region close
tothemiddleofthetest.
The DL (or least detectable dose) is the smallest
concentrationoftheanalytethatproducesasignalwhich
can be significantly distinguished from zero for a given
sample matrix with a stated degree of confidence. Very
oftenadoseisselectedwhichinhibits10–20%ofenzyme
tracer from binding with the Ab or the dose calculated
after subtraction of two or three times the standard
deviationfromthemeanmeasurementsofthezerodose
signal..3/
Linearizationofthecalibrationcurveisusefulformany
purposes,forinstance,forthedirectcomparisonofcurves
if matrix effects are evaluated. Absorbance curves can
be normalized by converting the absorptions to %B=B
0
values.Thesecanbeexpressedastheratioofboundtracer
inthepresenceofhaptentoboundtracerintheabsenceof
haptenandliesbetween100%(DA ,theupperasymptote
0
of the curve) and 0% (DA , the lower asymptote)
Excess
(Figure3b).TheyarecalculatedbyEquation(3):
%B A(cid:0)A
D Excess (cid:2)100 .3/
B A (cid:0)A
0 0 Excess
Linearization can be obtained by various mathematical
transformations..47/ Usually, IAs are evaluated with
commercialIAprograms,oftenbasedonlogisticmodels
(cf.Rodgers.48/andDudleyetal..49/),e.g.four-parameter
modelsorthemoresimplelogit-logtransformation(two-
parameter model, Figure3c) which can also be carried
outwithacalculator(s),Equation(4):
%B %B=B
logit Dln 0 .4/
B 100(cid:0)%B=B
0 0
Figure3 EIA for the determination of atrazine using pAb.
4.2 QualityControl (a)Absorption curve (means of three determinations(cid:6)stan-
darddeviations),(b)B=B curve,and(c)logit/logtransforma-
Precision and accuracy of IA are important properties 0
tionbythetwo-parameterfit.
whichdeservespecialattention.Thequalityandstability
of the employed material (microtiter plates, pipettes)
andreagents(e.g.Abs,enzymetracerorbuffers),playa Thereproducibilityistheabilitytoyieldthesameresults
crucialrole..50/ Thelong-termstabilityofreagentshasto within analyses, between analyses, and between opera-
beensured,e.g.byfreeze-dryingofAbsand,ifnecessary, tors. The investigation of the variability of an IA gives
addition of stabilizing components to the test reagents valuable information about the consistency of the test.
suchastheenzymetracer..51/ Coefficients of variation (CV) of IA measurements are
In spite of the simple handling of the assays, expert usuallybetween10and20%foranoptimizedassay,.52;53/
knowledgeisrequired,especiallytorecognizeandremove although more precise results can be obtained..54;55/
incident errors. Therefore, IAs should be performed by Same-dayandday-to-dayCVofsampleshavebeendeter-
trainedpersonnel.Thedevelopmentofsimpleandrapid minedindifferentmatrices..53;56/ Interlaboratorytestsof
assays, such as dip-stick assays or immunofiltration tests the same IA as that carried out by Hock and the IA
reducestherequirementfortrainedusers,butonehasstill StudyGroup.57/ andHayesetal..58/ fortheinvestigation
to be aware of potential problems such as interferences of triazines help to evaluate the general applicability of
fromthesamplematrix. atest.However,severalconditionslikeexactdescription
TheprecisionofanIAisdefinedastheextenttowhich of the assay including calibration curves, DLs, cross-
replicate analyses of a sample agree with each other. reactivities, a working range close to the middle of the
8
PESTICIDES
test,enoughparallelmeasurements,etc.mustbemet(see 100
alsoAOAC(AssociationofOfficialAnalyticalChemists) )
%
criteria).Meanwhile,standardizedproceduresforIAsin ( 80

y
waterareadoptedbyAOACInternationalandhavebeen t
vi
establishedinGermanyasaprenorm..50;58/ ti 60
c
A validation of the results obtained by IA should a
e
be carried out. To a limited extent this can be done r 40
-
s
by IA itself. Dilution of the samples as well as spiking s
o
of the authentic sample with known amounts of the r 20
C
contaminant can be used to check whether the matrix
interferes with the IA..59/ However, spiked samples do 0
1 2 3 4 5 1 2 3 4 5 1 2 3 4 5
not completely mimic real unknown samples. They do Selective Intermediate Group-specific
not contain potential metabolites of the contaminant
nor residues from other compounds which may be Figure4 Selective,intermediateandgroupspecificAbs.This
present in real samples. Furthermore, spiked samples exampleusesAbswhichhavebeenproducedagainsthapten1.
Substances 2–5 are assumed to be cross-reacting haptens
cannot be a model for aged residues which are more
(modifiedafterHocketal..14/).
difficult to extract and detect because, for example,
they may have bound to soil constituents. Therefore, an
IA should also be validated by a different established Strong cross-reactivities of an Ab to unexpected
method like high-performance liquid chromatography metabolites, for example, can produce false positive
(HPLC), gas chromatography (GC) or GC/MS (gas values. An Ab for alachlor was found to react very
chromatography/mass spectrometry).Manygroupshave stronglytothesulfonicacidmetaboliteusinganalachlor
usedthisapproachandhaveusuallyobtainedcorrelation screeningkit..69/ Thisproblemcouldbesolved,however,
coefficients of >0.9..60–63/ Often a slight overestimation by using solid-phase extraction (SPE) prior to IA and
of the IA in comparison with HPLC or GC is observed sequential elution of the two compounds with different
owingtocross-reactivitiesoftheAbormatrixeffects. organicsolvents.
4.3 Cross-reactivity 4.4 SamplePreparationandMatrixEffects
Depending on the conjugate used for immunization Samplescancontaincompoundsinadditiontothetarget
and the class of chemicals under investigation, cross- analyte,whichmayinterferewiththetest.Severalgroups
reactivities of the Ab with haptens similar to the investigatedtheinfluenceofionsonEIAs..70–72/Ruppert
analyte are frequently observed (see e.g. Hock.14/ and etal..70/ observed an inhibition by several anions like
Harrisonetal..64/).Therefore,itshouldbecheckedwhich azide, which inhibits the peroxidase by binding to the
compoundscross-reacttowhatdegreewiththeAb.This heme group of the enzyme. Most cations did not have
is usually done by comparing the standard curves of aneffectexceptforCa2C,whichleadstoanactivationof
the analyte under investigation with similar haptens, theperoxidase.Nointerferencebydifferentionssuchas
using analyte concentrations at 50% of the inhibition nitrate,copper,magnesiumetc.uptoaconcentration of
curve as the reference. However, cross-reactivity with a 250ppmwasdetectedinanEIAforpentachlorphenolin
certain analyte is not the same over the whole range water..71/ While ions may inhibit the enzyme used as a
of a standard curve. Often higher cross-reactivities can label or lead to precipitates by reacting with the buffer
be observed at low concentrations of the cross-reacting components, humic substances present in water or soil
analyte..3/ Therefore, it has been recommended that extractsmay bind nonspecifically to the Ab and thereby
cross-reactivitiesbemeasuredatdifferentconcentrations interferewiththespecificbindingoftheanalyte..73/These
overtherangewheretheassayissuitable..65/ reactionsmayleadtofalsepositiveresults.Watersamples
IfanAbisselectiveforasinglecompound,itisregarded from forest stands or soil extracts particularly contain
as monospecific.66/ (Figure4). An Ab that recognizes a high content of organic compounds such as humic
several compounds to the same extent (e.g. a group of acids(HAs).
s-triazines), can be used for the screening of a class Matrixeffectsinfoodsamplesfrequentlyoccurowing
of herbicides.67/ (group-specific Ab, Figure4). If cross- to colored extracts or to the content of lipids, proteins
reacting compounds are not expected in the samples, or polyphenols that may be coextracted during sample
becausethecompoundsarenotlicensed(e.g.propazinein preparation..74/ As food samples usually have to be
mostEuropeancountries),agroup-specificAbcanalsobe extractedpriortoimmunochemicalanalysis,themethod
usedforquantitativemeasurementsofonecompound..68/ of analyte extraction is of great importance. Analytes
9
IMMUNOCHEMICALASSAYSINPESTICIDEANALYSIS
that are water soluble and can be efficiently extracted methods in residue analysis. Not all of them are
in aqueous buffer will have the most direct extraction commercially available. Available commercial IAs have
method and eliminate the need for organic solvents. been listed in e.g. Dankwardt etal.,.2/ Hennion and
However, many pesticides are not readily water soluble Barcelo,.3/ Knopp,.8/ but a lot of movement has been
and must be extracted with an organic solvent..5/ For observed in environmental IA markets, leading to the
the extraction of pesticides from solid foods a variety disappearance of IA companies. At the moment IAs
of solvents have been tested, such as acetone, ether, for environmental contaminants can be obtained for
petroleum ether, methanol, acetonitrile or hexane..75/ example from Strategic Diagnostics Inc. (Newark, DE,
Direct analysis of extracts by IA requires the use USA, sells former Ensys, Millipore and Ohmicron kits)
of solvents that are miscible with water and (at low andEnviroLogix(Westbrook,MA,USA).
concentrations) are nondenaturing to proteins such as
Ab. IAs are to a certain degree tolerant to a variety of
5.1 Water
solvents, but each system must be tested to determine
which solvent can be accepted and to what extent (for EIA have been used intensively for the determina-
example Hill etal.,.75/ Nugent.76/ and Schneider and tion of pesticides in surface and rainwater.56;60;69;211–217/
Hammock.77/). Usually the extracts are further diluted and groundwater..60;69;214;218;219/ A substantial num-
with water prior to the EIA, but an EIA for parathion ber of these studies were carried out for triazine
wasdeveloped, inwhichtheanalytedissolvedinhexane herbicides..56;60;211;212;214;216;218;219/ This illustrates the
could be directly measured in the EIA without prior widespreadoccurrenceoftheseherbicidesintheaquatic
removal of the hexane. This was achieved by using Ab environment. Many groups have used commercial test
encapsulatedinreversemicellescomposedofAerosolT kits, which allow the investigation of samples without
withaqueouscenters..78/However,a104-folddecreasein time-consuming Ab production. Thurman etal.,.60/ for
sensitivitywasobserved. example,usedaRes-I-Munekit(ImmunoSystems)forthe
Insomecasesacleanupstepisintroduced,inwhichthe investigationoftriazinesinsurfaceandgroundwater.The
analyte of interested is separated from the matrix. This EIAwascomparedtoGC/MSresultsobtainedfromsam-
can be carried out by C -columns or immunoaffinity plesthatwereextractedbySPE.Correlationcoefficients
18
columns..69;79;80/ A very interesting approach is the between 0.91 and 0.95 were obtained after introducing
applicationofsupercriticalfluidextraction(SFE)priorto cross-reactivity factors for each of the triazines in order
immunoanalysis. These methods generally employ CO to calculate a sum parameter for the GC. The majority
2
orCO containingvariousmodifiers..5;81/ of the samples contained only atrazine (up to 3m gL(cid:0)1).
2
Some problems with interfering ions can be solved Therefore, the EIA results corresponded well with the
by changing the buffer of the assay system so that no atrazineconcentrationsobtainedbyGC/MS.
precipitates may be formed..70/ Addition of BSA to the Mouvet etal..220/ compared four commercially avail-
plates prior to the addition of the standard and sample able test kits and one in-house developed assay for the
solutions.82/ or to the enzyme tracer.73/ greatly reduces determination of triazines in surface and groundwater.
theinfluenceofhumicandfulvicacidsontheEIA.Itmay Operational characteristics, cross-reactivity, sensitivity,
alsobehelpfultoswitchtoadifferentbatchofAbsora CV and agreement with GC/LC (gas chromatogra-
different assay kit, as different Abs may show different phy/liquid chromatography) measurements were inves-
sensitivities to interfering substances. The buffering tigated. DLs were determined between 0.003 and
capacity of the assay buffer should also be checked, as 0.07m gL(cid:0)1.Intra-assayCVswerebelow7%foralltests,
somewaterorfoodsamplesmayshowrelativelylowpH interassayCVsbelow 20%.Correlation studiesbetween
values.Noeffects,however,wereobservedbetweenpH3 the EIA kits and GC/LC were carried out for samples
and10bydifferentinvestigators..56;67;83/ from different water matrices. Depending on the water
source,differentlevelsofsignificancewereobservedwith
differenttests.Thebestresultswereobtainedforsurface
water, while not all kits showed a good agreement for
5 APPLICATIONTOENVIRONMENTAL
lysimetersamples.
SAMPLES
Apart from the triazines some other pesticides were
investigatedinwatersamples,alsousingcommercialtest
IAs have been developed for many environmental kits. Alachlor was determined in ground and surface
contaminants during the 1990s. A list of several IAs water using commercial tests..213/ SPE was carried out
described in the literature can be found in Table4. priortoEIAtoremoveinterferingsubstancesandtocon-
Most of them have been developed in laboratories, centrate the analyte. Concentrations of up to 0.8m gL(cid:0)1
showing the increasing importance of immunochemical wereobserved,andacomparisonwithGC/MSshoweda
10
PESTICIDES
Table4 PesticideIAsdescribedintheliterature
Pesticides Test Ab Range,DL, Ref.
format ormiddleoftest(IC )
50
Herbicides
Alachlor EIA p 0.2–8m gL(cid:0)1 84
EIA p 0.1–10m gL(cid:0)1 85
EIA p,m 0.2–8m gL(cid:0)1 86
Amitrole EIA p 1.7–4200m gL(cid:0)1 87
Atrazine CLIA p 25–500ngL(cid:0)1 88
EIA p 0.5–10m gL(cid:0)1 89
EIA p 0.01m gL(cid:0)1(DL) 90
EIA m 0.03–1m gL(cid:0)1 35
EIA p 0.2–100m gL(cid:0)1 64
EIA p 0.011–33m gL(cid:0)1 91
EIA m 0.05–3m gL(cid:0)1 92
EIA p,m 0.1–100m gL(cid:0)1 93
EIA p 0.5–10m gL(cid:0)1 94
EIA m 0.05m gL(cid:0)1(DL) 95
EIA m 0.01–10m gL(cid:0)1 77
EIA p 0.03–3m gL(cid:0)1 96
EIA p 1–1000ngL(cid:0)1 34
Bentazon EIA p 2–24m gL(cid:0)1 97
Bromacil EIA p 0.1–160m gL(cid:0)1 98
EIA p 0.01–1m gL(cid:0)1 99
Chlorodiamino-s-triazine EIA p 160–480m gL(cid:0)1 100
Chlorsulfuron EIA p 0.1m gL(cid:0)1(DL) 101
Clomazone EIA p 2–250m gL(cid:0)1 102
EIA p 0.5–500m gL(cid:0)1 103
Cyanazine EIA p 0.035–3m gL(cid:0)1 104
EIA p 0.5m gL(cid:0)1(DL) 105
EIA p 0.5m gL(cid:0)1(DL) 106
Diethylatrazine EIA p 0.01–100m gL(cid:0)1 107
Diclofop-methyl EIA p 10–75m gL(cid:0)1 108
2,4-D EIA p 50–5000m gL(cid:0)1 109
EIA m 2–20m gL(cid:0)1 110
RIA p 0.1–10mgL(cid:0)1 111
EIA p 0.05–10mgL(cid:0)1 111
RIA p 5–250m gL(cid:0)1 112
PFIA m 0.6m gL(cid:0)1(DL) 113
RIA p 1–1000m gL(cid:0)1 114
EIA m 0.096m gL(cid:0)1(DL) 115
Dichlorprop PFIA p 0.01–100m gmL(cid:0)1 116
Diuron EIA m 2m gL(cid:0)1(IC ) 117
50
EIA p 0.05–1m gL(cid:0)1 118
Hexazinone EIA p 0.22–17.6m gL(cid:0)1 119
Hydroxyatrazine EIA m 0.03–1m gL(cid:0)1 120
EIA m 0.05m gL(cid:0)1(DL) 95
EIA p 0.01–10m gL(cid:0)1 66
EIA p 3–300m gL(cid:0)1 121
Imazamethabenz EIA p 0.5–32m gL(cid:0)1 122
Imazaquin EIA p 0.45–25m gL(cid:0)1 123
Isoproturon EIA p 0.01–10m gL(cid:0)1 124
EIA m 20–250m gL(cid:0)1 125
EIA NA 0.02–1m gL(cid:0)1 126
Maleichydrazide EIA m 0.01–11m gmL(cid:0)1 127
MCPB EIA p 0.03–0.9m gL(cid:0)1 128
Metazachlor EIA p 10–1000ngL(cid:0)1 129
(continuedoverleaf)